1. Field of the Invention
This invention relates to an improved method and reagent composition for determining magnesium levels in fluids, and more particularly to a colorimetric method which is performed directly on serum and other fluids without deproteinization.
2. Nature and Significance of Accurate Magnesium Determinations
Magnesium is extremely significant in human physiology. It is one of the most abundant cations in the body and, next to potassium, it is the most prevalent intracellular ion. Magnesium in its ionic form is essential to many physiological processes. It plays a vital and major role in carbohydrate and lipid metabolism by serving as an activator of adenosine triphosphate (ATP) in the transfer of energy rich phosphate. It is also essential as an activating ion for many enzymes involved in lipid, carbohydrate and protein metabolism. In muscle tissue, magnesium has a significant influence on neuromuscular apparatus. Magnesium ions are also essential for the preservation of the macromolecular structure of DNA, RNA and ribosomes, and in addition they play a vital part in bone formation and the maintenance of osmotic pressure.
The amount of magnesium in the body is particularly significant. Decreased levels of magnesium in the body produce muscle irritability which, if not corrected, can result in tetany (prolonged involuntary muscle spasms), which is clinically indistinguishable from that caused by decreased calcium levels, and convulsions. On the other hand, increased levels of magnesium have a curare-like effect, resulting in a loss of deep tendon reflexes, a loss of touch, temperature and pain sensation, respiratory failure, cardiac arrest.
Because of the vitally important roles magnesium plays in the normal functioning of life processes, it has long been recognized that it is necessary to be able to accurately and reliably measure magnesium levels in the body in order to aid the physician in diagnosis and treatment. In addition, it is correspondingly necessary that such results are able to be produced urgently in response to an emergency or STAT request from a physician. The ever-increasing recognition by clinicians of the need for frequent determinations of serum magnesium levels requires that the procedure desirably be capable of being performed by automated means. Of further importance, since blood samples obtained from pediatric or geriatric patients are usually very small, it is necessary that methods used for accurately determining magnesium levels desirably utilize no more than an extremely small sample volume.
3. Description of Prior Art
For many years, physicians regarded the determination of magnesium levels in serum or plasma to be of limited value due to the numerous errors to which the earlier techniques were subject, causing an unreliability which the prior art sought to avoid by several different approaches. In fact, even though improvements in certain respects were achieved, the great variety of methods still in current use for measuring the amount of magnesium in biological fluids is testament to the fact that none of them is completely satisfactory, even after long years of attempted improvement. Most are tedious, inaccurate or rely upon expensive instrumentation of limited usefulness which is not likely to be available except in the largest and most highly sophisticated clinical laboratories. Many difficulties have hindered the development of accurate and precise methods for the determination of magnesium, among which are the nonspecific nature of its precipitation reactions, the great liability to interference from other ions, and the relatively low intensity of its spectral lines.
Recently, direct colorimetric dye-complexing methods using indicators such as Magon, methylthymol blue, and calmagite have become increasingly popular. The direct dye-complexing methods which might be considered to be the most closely related of the prior art to the present method are those employing the dyestuff calmagite (3-hydroxy-4-[(2-hydroxy-5-methylphenyl)azo]-1-naphthalene-sulfonic acid). These methods involve the fact that calmagite is known to become "metallized" by reaction with several metal ions such as calcium, magnesium, iron, and copper to form a metallized complex which is colored and may be optically measured. The prior art has included EGTA (ethyleneglycol-bis[.beta.-aminoethyl ether]-N,N'-tetra-acetic acid) and potassium cyanide to mask the metallizing of calmagite by metals other than magnesium, such as iron, copper, and calcium, which are normally found in blood serum and which might otherwise interfere in the assay. Thus magnesium is the primary metal in blood serum which metallizes or binds with calmagite in the prior art procedures. Unmetallized calmagite is blue in an alkaline medium, and when metallized or bound with magnesium, calmagite forms a reddish-colored complex. The blue, unmetallized calmagite compound, therefore, has a different spectral absorbance peak than does the metallized or magnesium bound compound.
The prior art method of Gindler (U.S. Pat. No. 3,754,864 issued Aug. 28, 1973) and Gindler et al. (Clin. Chem. 17, 1971, p. 662) incorporates those known properties of calmagite and its reaction with metal ions and also those known properties of EGTA and potassium cyanide to mask the metallizing of calmagite by iron, copper, and calcium normally found in blood; but the Gindler art also teaches that protein in serum produces a spectral shift in the absorbance of free and metallized calmagite, thus introducing errors when a non-protein, i.e. aqueous, solution is used for calibration. The unmetallized calmagite, which is blue in an alkaline medium (having an optimal absorbance between 600 and 650 nm) binds with magnesium to form a reddish-colored complex having an optimal absorbance of about 535 nm. The prior art teaches that the absorbance measurement at 532 nm is proportional to the amount of magnesium and thus serves as a quantitation of the amount of magnesium.
The Gindler prior art teaches that the spectral absorbance peak of the reddish-colored metallized dye is shifted in the presence of protein from 535 to 540 nm. In order to overcome this alleged interference from protein, selected micelle-forming protective colloids are added to the reaction mixture to mimic the interference from protein. These added substances, the prior art asserts, achieve spectral correlation between protein-containing and aqueous samples. Specifically, the prior art teaches that the absorbance peak of both protein-containing and aqueous samples is shifted to 545 nm when a micelle-forming protective colloid is added. Although this prior art method does achieve certain advantages over the earlier art, there remain or are created certain drawbacks, as now discussed.
The working reagent mixture of the Gindler et al. prior art is stable for no longer than several hours, thereby requiring not only the labor, cost and effort of reagent preparation every day, but the disposal and costly wastage of unused, prepared working reagent at the end of the day, and inaccuracy or unworkability if attempted to be used after that short stability period.
The addition of a micelle-forming protective colloid is believed, according to the novel concepts of the present invention, to unnecessarily complicate the formulation and possibly introduces adverse properties to the assay. The prior art teaches a measurement at 532 nm (even though it teaches a spectral shift to 545 nm), which is close to the absorbance of chromogenic substances such as hemoglobin, bilirubin, and lipemia which may be present in certain patients' serum. Consequently, when measurements are made at 532 nm on serum which contains hemoglobin, bilirubin or which is lipemic, the absorbance of these substances is likely to be wrongly determined as being due to the presence of magnesium. Therefore, the analyst could be led to report a higher level of magnesium to be present in the patient's blood serum than is actually present, thereby potentially leading the physician to a mistaken diagnosis or improper treatment of the patient, with obviously dangerous or other disadvantageous results.
Furthermore, the normal range of serum magnesium levels is a very narrow one, i.e., ranging from only 1.7 to 2.1 milliequivalents per liter of serum, and it is readily apparent that a clinically useful assay for magnesium must provide sufficient accuracy and sensitivity to accurately distinguish between normal and pathological levels. The limited sensitivity of the prior art methods for magnesium measurement diminish the likelihood of precise measurements of serum magnesium levels.
More recently, Denney et al. in co-pending U.S. patent application (Ser. No. 282,721, filed July 13, 1981), now U.S. Pat. No. 4,383,043, issued on May 10, 1983, the content of which is hereby incorporated by reference, describe an improved colorimetric assay method and reagent which help to solve or minimize many of the problems which plagued the prior art methods described above. The present invention provides even further improvement and/or an alternative means for improving upon these prior art methods.